PROJECT SUMMARY Mammalian genomes harbor three RAS genes, HRAS, KRAS and NRAS, which are mutated in cancer more frequently than any other oncogene. RAS proteins are small GTPases that function as binary switches to regulate a wide array of signaling pathways. RAS proteins propagate signals only when associated with cellular membranes as a consequence of post-translational modification of a C-terminal CAAX sequence. Farnesyltransferase (FTase) adds a polyisoprene lipid to the CAAX cysteine. RAS converting enzyme 1 (RCE1) removes the AAX amino acids. The newly C-terminal farnesylcysteine is then methylesterified by isoprenylcysteine carboxyl methyltransferase (ICMT). Each of the three CAAX processing enzymes is a potential target for anti-RAS drug discovery. We cloned mammalian ICMT 19 years ago, characterized the enzyme and its role in RAS biology and developed inhibitors. In the last cycle of this grant we elucidated the mechanism whereby genetic ablation of ICMT paradoxically exacerbated a mouse model of KRAS-driven pancreatic neoplasia and showed that this phenomenon was related to NOTCH signaling and was highly context-dependent in that ICMT inhibition inhibited the growth of other tumors. We also discovered that among the four RAS proteins, NRAS is uniquely sensitive to ICMT deficiency. Not only was NRAS completely blocked from the plasma membrane, but the cellular levels of the NRAS proteins were significantly decreased. Mutant NRAS drives hematopoietic malignancies and melanoma. Based on our exciting preliminary data we hypothesize that ICMT inhibition may be an attractive therapeutic modality for NRAS mutant melanoma, an important unmet clinical need. In the next cycle of this grant we will take advantage of CRISPR/Cas9 gene editing and the recent availability of a specific, nanomolar ICMT inhibitor to test this hypothesis with three specific aims. Aim 1. Role of ICMT in NRAS trafficking and signaling. We will study the effect of ICMT deficiency on NRAS post-translational modification, membrane trafficking, interactome and signaling in melanoma cells including short-term cultures. Aim 2. Role of ICMT in NRAS expression. We will determine the mechanism whereby ICMT deficiency leads to decreased expression of NRAS. Aim 3. Role of ICMT in growth and survival of NRAS-driven melanoma. We will study the proliferation and survival of melanoma cells (including short-term cultures) ICMT in 2D and 3D culture, in soft agar and in xenografts, we will cross our conditional ICMTfl/fl mouse with a model of NRAS-driven melanoma to determine if ICMT inhibition is protective in vivo, and we will determine if ICMT deficiency in lymphocytes alters immunogenicity of melanomas. Successful prosecution of these aims will define the degree and mechanisms whereby NRAS-mutant melanoma cells are dependent on ICMT and thereby provide the preliminary data required for clinical trials of ICMT inhibitors.